World Small Animal Veterinary Association World Congress Proceedings, 2013

Mike Shipstone, BVSc, FANZCVS, DACVD

Dermatology for Animals, Stafford Heights, QLD; School of Veterinary Science, University of Queensland, QLD, Australia

There is a range of diagnostic procedures that can easily be conducted in a consult room which, whilst requiring minimal equipment, give an enormous amount of information in the management of dermatology cases.

Microscope Setup

There are 2 basic cytology samples that can be examined using a microscope. Those that require some form of stain to identify the organism of interest and those that are unstained. Each requires the microscope to be set up in a different way that allows the easy identification of the target organisms.

1. Unstained cytology
This may be used to examine the hair shaft or look for various mites. The microscope must be set to increase the natural contrast of the sample. This can be achieved in a number of ways:

a. Using phase contrast if the microscope has this facility.

b. Phase can be mimicked by closing the iris on the condenser or by lowering the condenser away from the microscope stage.
The sample is placed on a glass slide and a coverslip is placed over the top, which has the effect of creating an even thickness to the sample and therefore an even plane of focus for examination.

2. Stained cytology
This is used to examine all other types of cytology whether the target of interest is some microorganism, an inflammatory cell or fine-needle aspirate. The material collected has no colour and cannot be seen until a range of stains is used to highlight it. In contrast to the previous cytology, the microscope is set up with the iris open, the condenser level with the bottom of the microscope stage or the phase contrast disengaged.

Diagnostic Procedures

Unstained Cytology

1. Coat Combing

The coat is brushed with a flea comb and the resultant scurf examined for surface parasite identification. With some parasites (particularly Cheyletiella), concentration of the scurf via emulsion in faecal float solution may be necessary.

Parasites identified include:

Cheyletiella

Lice

Fleas

Trombicula

2. Skin Scraping

Superficial

The area to be sampled is lightly clipped, paraffin oil is placed on the skin and a blunt scalpel is used to collect surface scale and crust. The aim is not to cause capillary bleeding as the parasites being collected are in the superficial layers of the skin.

Once the material is collected onto glass slides, it is covered with a coverslip (to make an even plane of focus), and the condenser is lowered or the iris closed (to increase the contrast) so that a normal microscope behaves like a phase contrast microscope.

The following parasites may be identified:

Sarcoptes

Cheyletiella

Demodex cornei

Demodex gatoi

Trombicula

Lynxacarus radovsky

Dermanyssus gallinae

Deep

This involves a deeper level of scrape - until capillary oozing is seen. This is done whenever Demodex is suspected. But as there is no such thing as a 'classic' look to demodicosis, it should be a routine test method in most investigations.

The area to be sampled is clipped (#40 blade) and then the skin is squeezed (increases mite yields), paraffin oil is applied and a blunt scalpel is used to scrape until capillary oozing (i.e., not bleeding from laceration) is evident. The material is transferred to a glass slide and examined as above. NB the feet are often difficult to scrape and will bleed before an adequate depth is reached. Hair plucks may be performed and the base of the hair examined for the mites. If scraping the feet, move to an erythematous area at the edge of the affected area rather than the more swollen, severely affected skin. If the scrapes are negative, a biopsy is sometimes necessary to establish the diagnosis (this is also true for the Shar Pei).

3. Trichogram

A very simple yet powerful technique that allows the following to be assessed:

Anagen:telogen ratio

Congenital defects

Infections

Self-trauma

Follicular casts

Pigment disturbances

Hair is grasped with either your fingernails or padded mosquito forceps and placed in a drop of oil on a glass slide. A coverslip is placed on top and the hair examined with the microscope set up as above to increase contrast.

Stained Cytology

One of the most powerful investigative techniques is the collection and examination of cytology samples. The following may be identified:

Bacteria

Malassezia

Fungal elements

Inflammatory cells

Acantholytic cells

There are a number of different techniques of collection each useful in its own situation. No one method is better than another, and several may be used in different areas on the same animal.

1. Acetate Tape Preparations

Particularly where lesions are dry/flaky

Interdigital areas

Periocularly

The acetate tape is placed onto the skin surface a number of times in the same place (6 times will remove the entire stratum corneum). It is then placed sticky side down onto a glass slide and stained with the purple stain of a modified Wright's stain (e.g., Diff-Quick). The microscope is set up so that the iris is completely open with the condenser set high. The epithelial debris and any organisms can be clearly seen and emersion oil can be applied to the back of the tape if required (without the need of a coverslip).

NB the tape used must be the old-fashioned clear tape (i.e., not the frosted "magic" or invisible tape).

The material collected with the following techniques is all stained in a similar manner. The material is air dried and may be heat-fixed if necessary. It is then stained with Diff-Quick and examined with the microscope set up as for acetate preparations. Gram stains may also be performed.

2. Direct Smear

Fluid-containing lesions

3. Impression Smear

Moist/greasy lesions

After crust removed

Expressing fluid from lesion

Opening surface of pustule/papule/vesicle

4. Cotton Bud Smear

Draining tracts

Ear canals

Interdigital

5. Scalpel Blade

Sample under crust

Vesicles

Peeling St. corneum

6. Fine-Needle Aspirate

Nodules, tumours, cysts

A 22-ga needle with a 12-ml syringe is used to collect material from the centre of the lesion. The material is aspirated onto a slide, dried and stained as above. Neoplastic cells may be difficult to identify so the help of a clinical pathologist should be sought. Even with high skill levels, tumours should alwaysbe diagnosed on the basis of histopathology.

The hair shafts that fluoresce may be examined for the presence of arthrospores (fungal elements) at the bulb of the hair. Most veterinary dermatophytes are ectothrix invaders. Examination of the hairs may require chlorphenolac or potassium hydroxide clearing to allow better visualisation of the elements.

3. Fungal Culture

NB only for dermatophytes; if deep mycoses or other fungal infections are suspected, these should be sent to an accredited laboratory for culture because of the occupational health and safety risks.

Culture medium

Dermatophyte test medium (DTM)

Observe daily

Dermatophytes

Metabolise protein alkaline colour change

Blastomyces, Sporothrix schenckii, Histoplasma capsulatum, Coccidioides, Pseudoallescheria, some Aspergillus species may cause a change to red in DTM

Occasional saprophyte (Scopulariopsis) can false positive

Microscopic examination is essential

Cryptococcus, Candida, Aspergillus and many agents of phaeohyphomycoses are inhibited by cycloheximide 6

Saprophytes

Metabolise CHO acid no colour change initially, but once culture is large, there is a sudden (overnight) colour change of the entire test media.

Sabouraud's dextrose agar

This is used to allow the fungus to form macroconidia more efficiently and allow species identification.

Used in the following circumstances:

Granulomatous lesions, draining tracts

Non/poor response to appropriate antibiotics

Rods present on cytology

Biopsy

A biopsy should be collected for any of the following circumstances:

Neoplasia/suspected neoplasia

Persistent ulceration

Failure to respond to previous treatment

Unusual/serious clinical signs

Vesicular dermatosis

Treatment is expensive and/or potentially dangerous

In general a biopsy should be collected within 3 weeks if a dermatosis is not responding to what appears to be appropriate therapy. Biopsy collection early in the disease process helps avoid nonspecific or masking changes as a result of chronicity. Lesions evolve through a life cycle from early to mature to chronic. The most diagnostic stage is generally early in the process. Chronic changes are commonly nonspecific and nondiagnostic. Early biopsy and definitive diagnosis also allow appropriate therapy to be started.

Site Selection

Selection involves careful examination of the entire animal for the most representative lesions. They should cover the range of spectrum from very early to more mature, but should include the active stages. Including the spectrum of lesions gives far more information and greatly enhances the chance of a diagnosis. For example, if investigating depigmentation, it is essential to biopsy the actively depigmenting lesions (i.e., the grey areas, not the chronic pink areas where a diagnosis is much less likely). Alopecia should be sampled from the centre, junctional and normal skin. Multiple samples should be selected, as this increases the chance of a diagnosis.

Site Preparation

There should be no surgical preparation. Do not scrub or clean the skin in any way as this will remove the surface layers which are often critical for diagnosis.

The only preparation is the gentle removal of hair.

If the lesion is crusted and this crust is lost during sampling, include it in the formalin pot so that it can be cut in. Remember to advise the pathologist that the crust has been included so that it is added to the block for sectioning.

Biopsy Technique

1. Wedge

a. Common for excisional collection of nodules or tumours

b. Vesicular lesions

c. Panniculitis

d. Edge of lesion (allows orientation by the pathologist). Traditionally the lesion is biopsied so that the spectrum, from normal to affected, follows the long axis of the sample. This means that the sample can be sectioned in this plane and represents the graduation of pathology as well.

2. Punch

This is the most commonly used technique. Generally a 6-mm or 8-mm punch is used for most areas, with a smaller (4-mm) punch used for the nose, lips or mouth. The area is clipped of hair and local anaesthetic instilled under the site. The punch is placed firmly on the skin and rotated in one direction whilst continuing to push. This will allow sampling of the entire skin thickness down to the subcutaneous tissue. Fine scissors are used to separate the sample from the subcutis. The tissue is blotted free of blood and placed on a piece of paper and then dropped into the formalin. Placing the sample on a piece of stiff paper or tongue depressor prevents it from curling when placed in the formalin. This is particularly important when collecting thin skin.

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